Xenon-129 biosensors offer an attractive alternative to conventional MRI contrast agents due to the chemical shift sensitivity and large nuclear magnetic resonance signal of hyperpolarized 129Xe. Here we report the use of fluorescence spectroscopy and isothermal titration calorimetry (ITC) to determine xenon binding affinity and thermodynamics with a water-soluble triacid-cryptophane-A (1). 1 was synthesized in 10 steps with a 4% overall yield. Fluorescence spectroscopy measured an association constant of (1.7 ± 0.2) × 104 M-1 in phosphate buffer at 293 K. ITC measurements at 293 and 310 K yielded association constants of (1.73 ± 0.17) × 104 and (3.01 ± 0.26) × 104 M-1 and indicated a large entropic contribution to xenon binding in water. On the basis of these data, cryptophane 1 showed roughly 2-fold higher affinity for xenon than any previously measured compound. Remarkably, ITC measurements in human plasma at 310 K gave a similar binding constant, K A = (2.19 ± 0.22) × 104 M-1, which supports the development of 129Xe NMR biosensors for biological applications.

Xenon-129 biosensors offer an attractive alternative to conventional MRI contrast agents due to the chemical shift sensitivity and large nuclear magnetic signal of hyperpolarized (129)Xe. Here, we report the first enzyme-responsive (129)Xe NMR biosensor. This compound was synthesized in 13 steps by attaching the consensus peptide substrate for matrix metalloproteinase-7 (MMP-7), an enzyme that is upregulated in many cancers, to the xenon-binding organic cage, cryptophane-A. The final coupling step was achieved on solid support in 80-92% yield via a copper (I)-catalyzed [3+2] cycloaddition. In vitro enzymatic cleavage assays were monitored by HPLC and fluorescence spectroscopy. The biosensor was determined to be an excellent substrate for MMP-7 (K(M) = 43 microM, V(max) = 1.3 x 10(-)(8) M s(-1), k(cat)/K(M) = 7,200 M(-1) s(-1)). Enzymatic cleavage of the tryptophan-containing peptide led to a dramatic decrease in Trp fluorescence, lambda(max) = 358 nm. Stern-Volmer analysis gave an association constant of 9000 +/- 1,000 M(-1) at 298 K between the cage and Trp-containing hexapeptide under enzymatic assay conditions. Most promisingly, (129)Xe NMR spectroscopy distinguished between the intact and cleaved biosensors with a 0.5 ppm difference in chemical shift. This difference most likely reflected a change in the electrostatic environment of (129)Xe, caused by the cleavage of three positively charged residues from the C-terminus. This work provides guidelines for the design and application of new enzyme-responsive (129)Xe NMR biosensors.

Cryptophane-A, comprised of two cyclotriguaiacylenes joined by three ethylene linkers, is a prototypal organic host molecule that binds reversibly to neutral small molecules via London forces. Of note are trifunctionalized, water-soluble cryptophane-A derivatives, which exhibit exceptional affinity for xenon in aqueous solution. In this paper, we report high-resolution X-ray structures of cryptophane-A and trifunctionalized derivatives in crown–crown and crown–saddle conformations, as well as in complexes with water, methanol, xenon or chloroform. Cryptophane internal volume varied by more than 20% across this series, which exemplifies 'induced fit' in a model host–guest system.

A water-soluble triacetic acid cryptophane-A derivative (TAAC) was synthesized and determined by isothermal titration calorimetry (ITC) and fluorescence quenching assay to have a xenon association constant of 33,000 M−1 at 293 K, which is the largest value measured for any host molecule to date. Fluorescence lifetime measurements of TAAC in the presence of varying amounts of xenon indicated static quenching by the encapsulated xenon and the presence of a second non-xenon-binding conformer in solution. Acid-base titrations and aqueous NMR spectroscopy of TAAC and a previously synthesized tri-(triazole propionic acid) cryptophane-A derivative (TTPC) showed how solvation of the carboxylate anions can affect the aqueous behavior of the large, nonpolar cryptophane. Specifically, whereas only the crown-crown (CC) conformer of TTPC was observed, a crown-saddle (CS) conformer of TAAC was also detected in aqueous solution.

The (13)C NMR spectra of two different iodoalkynes, 1-iodo-1-hexyne (1) and diiodoethyne (2), exhibit a strong solvent dependence. Comparisons of the data with several common empirical models, including Gutmann's Donor numbers, Reichardt's E(N)(T), and Taft and Kamlet's beta and pi, demonstrate that this solvent effect arises from a specific acid-base interaction. Solvent basicity measures such as Donor numbers and beta values correlate well with the alpha-carbon chemical shift of 1, but polarity measures such as E(N)(T) and pi do not correlate. The similarity of the solvent effect for 1 and 2 suggests that carbon-carbon bond polarization may not play a role in the change in chemical shift, as previously hypothesized.

Hyperpolarized 129Xe chemical exchange saturation transfer (129Xe Hyper-CEST) NMR is a powerful technique for the ultrasensitive, indirect detection of Xe host molecules (e.g., cryptophane-A). Irradiation at the appropriate Xe-cryptophane resonant radio frequency results in relaxation of the bound hyperpolarized 129Xe and rapid accumulation of depolarized 129Xe in bulk solution. The cryptophane effectively ‘catalyzes’ this process by providing a unique molecular environment for spin depolarization to occur, while allowing xenon exchange with the bulk solution during the hyperpolarized lifetime (T1 ≈ 1 min). Following this scheme, a triacetic acid cryptophane-A derivative (TAAC) was indirectly detected at 1.4 picomolar concentration at 320 K in aqueous solution, which is the record for a single-unit xenon host. To investigate this sensitivity enhancement, the xenon binding kinetics of TAAC in water was studied by NMR exchange lifetime measurement. At 297 K, kon ≈ 1.5 × 106 M−1s−1 and koff = 45 s−1, which represent the fastest Xe association and dissociation rates measured for a high-affinity, water-soluble xenon host molecule near rt. NMR linewidth measurements provided similar exchange rates at rt, which we assign to solvent-Xe exchange in TAAC. At 320 K, koff was estimated to be 1.1 × 103 s−1. In Hyper-CEST NMR experiments, the rate of 129Xe depolarization achieved by 14 pM TAAC in the presence of RF pulses was calculated to be 0.17 µM·s−1. On a per cryptophane basis, this equates to 1.2 × 104 129Xe atoms s−1 (or 4.6 × 104 Xe atoms s−1, all Xe isotopes), which is more than an order of magnitude faster than koff, the directly measurable Xe-TAAC exchange rate. This compels us to consider multiple Xe exchange processes for cryptophane-mediated bulk 129Xe depolarization, which provide at least 107-fold sensitivity enhancements over directly detected hyperpolarized 129Xe NMR signals.

Efficient syntheses of trisubstituted cryptophane-A derivatives that are versatile host molecules for many applications are reported. Trihydroxy cryptophane was synthesized in six or seven steps with yields as high as 9.5%. By a different route, trihydroxy cryptophane modified with three propargyl, allyl, or benzyl protecting groups was synthesized with yields of 4.1-5.8% in just six steps. Hyperpolarized 129 Xe NMR chemical shifts of 57-65 ppm were measured for these trisubstituted cryptophanes.Cryptophane organic host molecules, constructed from two cyclotriguaiacylene (CTG) units connected by three alkane linkers, possess a hydrophobic cavity that can encapsulate a wide variety of guests. One important application involves xenon binding to cryptophane, which can be delivered to specific cellular targets for detection and resolution by 129 Xe magnetic resonance spectroscopy or imaging. 1 Currently, water-soluble cryptophane-A derivatives show the highest known xenon affinity with K A ≈ 30,000 M −1 in buffer at rt. 2 129 Xe can be hyperpolarized to generate ~10 5 NMR signal enhancements and provides a greater than 200 ppm 129 Xe NMR chemical shift window, with resonance frequencies that depend sensitively on the molecular environment. 3 Thus, cryptophane hosts functionalized with different recognition moieties allow the simultaneous detection of multiple targets (i.e., multiplexing), as is desirable for biomolecular imaging. 4 The importance of in vivo studies has motivated the development of synthetic routes capable of producing large quantities of functionalized cryptophane. 5 A previously described multi-step template strategy allowed the synthesis of diverse mono6 and tri-functionalized cryptophane-A derivatives2 ,7 as well as enantiopure (−)-cryptophane-A.8 However, even improved synthetic routes typically involve nine or more steps with low yields.5b The preparation of separate connecting linkers and CTG units is time-consuming, and the hydroxyl functionalities must be protected to avoid side-products during the cryptophane synthesis. Moreover, the two cyclization reactions to produce first CTG and finally cryptophane typically involve strong acid such as perchloric acid in methanol or formic acid.9 These conditions are incompatible with the synthesis of new CTG derivatives bearing acid-sensitive moieties, and very often dilute conditions are required to avoid polymerization, as in the case with propargyl groups.2 Recently, Brotin and coworkers * Corresponding author: Dmochowski, Ivan J., ivandmo@sas.upenn.edu. Supporting Information Available:Experimental procedures and characterization data for all synthesized compounds and 129 Xe NMR data. This material is available free of charge via the Internet at http://pubs.acs.org. reported cyclization reaction conditions using a milder reagent as Lewis acid, Sc(OTf) 3 . 10 Notably, in some cases even better yields were obtained, and the purification steps were made easier. NIH Public AccessBased on these observations, we developed a shorter, 6-step synt...

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